Rea Lec 2 Microscopy FP

8/13/2019 Rea Lec 2 Microscopy FP

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Visualizing Cells


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Learning Objectives

History of Microscopy (Hooke, Zeiss, Perkin)Anatomy of Compound Microscope Capabilities & Limitations of Various Technologies Fluorescence Microscopy Laser Scanning Microscopy Fluorescent Proteins


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Early Cell Biologists


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Robert Hooke (1635 - 1703)

Built one of the first useful compound microscopesObserved structure of cork

Coined the term Cell.

Published Micrographia(1665)


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1665 Hooke publishes Micrographia

1678 van Leeuwenhoek observes protozoa (little animals)

1838-9 Schleiden & Schwann proposed Cell Theory

1857 Carl Zeiss produces the Stand 1microscope

1865 Perkins invents synthetic (aniline) dyes

A Brief History of Optical Microscopy


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Stand 1MicroscopeCarl Zeiss

(1816 1888)

Microscopy for the Rest of Us


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stem section mouse fibroblast


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1665 Hooke publishes Micrographia

1678 van Leeuwenhoek observes protozoa (little animals)

1838-9 Schleiden & Schwann proposed Cell Theory

1857 Carl Zeiss produces the Stand 1microscope

1865 Perkins invents synthetic (aniline) dyes



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William Henry Perkin(1838 1907)

August Wilhelm von Hoffman(1818 1892)

Pioneers of Organic Synthesis


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Quinine

quina-quinaChichona calisaya

Early Anti-malarial Drugs


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3-amino-2,9-dimethyl-5-phenyl-7-(p-tolylamino)phenazinium acetate

Mauveine (Perkins Mauve)


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(spiny dye-murex snail)Justinian I(482 - 565)

Bolinus brandaris

Imperial (Tyrian) Purple


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Figure 1-5 Essential Cell Biology ( Garland Science 2010)


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Walther Flemming(1843 1905)

Zellsubstanz, Kern

und Zelltheilung, 1882

Discovery of Chromosomes (Colored Bodies) & Mitosis


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1931 Ruska invents electron microscope

1932 Zerniki develops phase contrast microscopy

1955 Minsky invents the laser scanning microscope (LSM)

1989 Webb, Denk & Strickler invent multiphoton LSM

1995 Stefan Hell invents Super Resolution Microscopy



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Compound Microscope *


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Detection & Analysis

Magnification

Light Gathering

Illumination

*


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Olympus IX71

Inverted MicroscopeOlympus BX51

Upright Microscope


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Issac Newton

Albert Einstein Richard FeynmanJames C. Maxwell

George B. AiryAlhazen


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James C. Maxwell

Electromagnetic Theory of Light Propagation

*


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Wave-like Properties of Light

refraction diffraction

*


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Refraction at the Air - Water Interface


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Optical Convergence using a Thin Lens


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n1sin!= n2sin"

n2 sin!

=

n1 sin"

For n2> n1

nair = 1.0

n2 =sin!

sin"

water

air

nwater = 1.33

nglass = 1.51

Snells Law *


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Ripple Tank


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Diffraction is a characteristic of wave dynamics

Slit ~ wavelength ($)

barrier

slitba

rrier


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Slit ~ 4 $


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Interference

Positive

Interference

Negative

Interference

*


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*


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*


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Phase Contrast Microscopy

*


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Bright Field Phase Contrast

Differential Interference

Contrast (DIC)

Nomarski

Dark Field

Images of a Fibroblast *


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Objective Lens capturesonly a small portion of

the light rays that are

diffracted and refracted

by the specimen.

The optical imageis

always incomplete


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(0.61)$

n sin ( )

$= wavelength of lightn = refractive index

= angular aperture

Limit of resolution (D)

D =

Limit of Spatial Resolution of Optical Microscopy

NOT A FUNCTION OF MAGNIFYING POWER OF LENS

*


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(0.61) $n sin ( )



D =

Numerical Aperture

(light gathering ability)

N.A. ~ 0.3 - 1.65


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(0.61) $n sin ( )



D =

Numerical Aperture

(light gathering ability)

N.A. ~ 0.3 - 1.65

Higher is Better

*


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Objective Lens


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(0.61)$

n sin ( )



D =

Wavelength of Light

Shorter is Better


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Visible Spectrum


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D =(0.61) $

n sin (

)

(0.61) (450 nm)

(1.5) sin (70o)D =

~ 200 nm

~ 0.2 m (~ /2)glass & oil

70o

Resolution is ultimately limited by the

wavelength of the illuminating light

$

Limit of Resolution approaches ~ !of the wavelength


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Electron Microscopy

Array of Au atoms

Wavelength ($) of an(accelerated) electron

= 0.004 nm

Resolution = $/ 2~ 0.002 nm

This is 100,000 times

better than optical

microscopy ~ 200 nm


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glass

glass

glass

retina

visible light

magnet

magnet

magnet

phosphor

electrons

Light Microscopy TEM


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Figure 9-54 Molecular Biology of the Cell( Garland Science 2008)

Actin Filaments


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Scanning electron microscopy (SEM)


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*


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SEM of a metal cast

of a wheat flower.

Its not so muchabout size --

Its about resolution


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SEM

DIC

TEM

Stereocillia from bullfrog auditory hair cell


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Fluorescence Microscopy

mouse fibroblast mouse fibroblast


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Stuff Fluoresces


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Fluorescence Spectrum of FITC

blue red

absorbanceemission

{

Stokes Shift

*


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Fluorescence Spectrum of FITC

fluorescein isothiocyanate(FITC) blue red

absorbanceemission

{

Stokes Shift


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% %

Sigma (% vs Pi (&) Bonding


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Filter CubeEnables Fluorescence Microscopy

Filter Cube


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Filter CubeEnables Fluorescence Microscopy


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Filter

Cube

*


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Indirect Immunofluorescence *


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All organic fluors undergo photobleaching


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Quantum Dots


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Quantum Dots

Highly resistant to photobleachingAll are excited by UV light

Emitted color is a function of size

*


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slit

Conventional Fluorescence Microscopy


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Conventional Fluorescence Microscopy


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Epifluorescence Image Confocal Image

All emitted light

under the objective

lens

Only light emitted

from within the focal

plane

More photons Better photons

*


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Confocal Imaging by laser scanning microscopy (LSM)

Confocal Microscope

Light

Source

Object plane

Objective Lens

Image plane

Tubus Lens

Light

Source

Object plane

Objective Lens

Image plane

Tubus Lens

Pinhole GalvanometerxScannerGalvanometer

yScanner

Laser

Objective

Lens

Object

Plane

Mirror

y

x

Galvanometer

xScannerGalvanometer

yScanner

Laser

Objective

Lens

Object

Plane

Mirror

y

x

Line Scan

laser


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y

z

x

z-Stack

50 mReconstructed

Neuron

Confocal Imaging

Confocal Microscope

Light

Source

Object plane

Objective Lens

Image plane

Tubus Lens

Light

Source

Object plane

Objective Lens

Image plane

Tubus Lens

Pinhole


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3D Reconstruction from Consecutive Optical Sections

Metaphase Spindle Complex


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Aequorea victoria


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Green Fluorescent Protein


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Green Fluorescent Protein

GFP Fluorophor

- Ser65 - Tyr66 - Gly67 -


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GFP transcriptional reporter


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GFP transcriptional reporter


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GFP variants produced by artificial selection in E coli.

Roger Tsien Lab


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Tsien, Shimomura, Chalfie2008 Nobel Prize in Chemistry

Roger Tsien


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Deconvolution Microscopy

Point Spread Function for 2 Fluorescent Dyes


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Deconvolution Microscopy

Acquired Image Deconvolved Image

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Rea Lec 2 Microscopy FP